Iron is necessary for the enzymatic activity of key proteins in the DNA synthetic pathway, electron transport in the oxidative phosphorylation pathway in mitochondria, the activity of enzymes in the citric acid cycle, and the activity of proteins in many other essential metabolic and catabolic pathways in cells. In the body, iron is bound to transferrin for transport through the serum. Transferrin, in turn, is taken up into cells by receptor- meditated endocytosis through binding to the transferrin receptor on the cell surface. The long term objective my research is to understand the structure and function of the transferrin receptor. This proposal focuses on the mechanism by which the receptor concentrates into clathrin-coated pits. The cytoplasmic domains of receptors and in particular four to six amino acid Tyr-based motifs are critical for the clustering of receptors in clathrin-coated pits. The Tyr-based motifs were originally thought to interact with a single protein in the coated pit. Recent evidence from several laboratories, however, indicates that a variety of receptors do not compete with each other during endocytosis. This proposal will test the model that receptors interact with different components of the endocytic apparatus according to the primary or tertiary structure of their cytoplasmic domains. Specific Aim 1 will determine whether structures in cytoplasmic domains define the ability of some receptors to compete with other receptors for endocytosis. Fluorescence-activated cell sorting analysis will be used to establish which receptors when overexpressed compete for endocytosis. Structural analysis of the cytoplasmic domains will be used to determine whether the structures of the cytoplasmic domain correlate with the competition studies. Specific Aim 2 will then determine whether competition is defined by interaction with the same connector protein in the endocytic apparatus. The yeast two-hybrid system will be used to identify proteins which interact with different classes of the cytoplasmic domains of these receptors. Candidate proteins will be tested in vitro and in vivo for their ability to interact with the cytoplasmic domains of receptors. Antibodies will be generated to these proteins to determine their subcellular localization. Endocytosis is essential for the uptake of not only iron and other nutrients into the cell but also for the clearance of cholesterol from the blood, the down regulation of signal transduction molecules, and the signal transduction process itself. Understanding this process will give insight into such diseases as hemochromatosis, hyperlipidemia, and virally-mediated down regulation of immune responses.